Protease-activated receptors (PARs) were discovered in the context of an effort to understand how thrombin and other proteases regulate cellular behaviors. Four PARs are found in mammals. PARs 1,3, and 4 are thrombin receptors that mediate activation of platelets and other cells by thrombin and play key roles in hemostasis and perhaps inflammatory and other responses to tissue injury. By contrast, physiological activators of PAR2 have not been clearly identified, and its roles in vivo are uncertain. Our recent work suggests that PARs play roles in contexts other than response to injury. PAR1 function in endothelial cells is important for proper vascular development, and we now find that PAR2 also plays distinctive roles in the embryo. In one strain background, Par2-/- embryos develop profound anemia. Moreover, Par1 -/-Par2 -/- embryos show a strong phenotype not seen in either single knockout: exencephaly, a hallmark of failed neural tube closure. PAR2 is expressed in the epidermal ectoderm overlying the neuroepithelium before and during neural tube closure, that is, at the right place and time to play a direct role. We will better define these interesting phenotypes and identify the exact cell types involved by transgene rescue and cell type-specific knockout. What does PAR2 regulate and what does it sense in these contexts? We have developed mouse lines that will be used to ablate specific G protein signaling pathways in the cell types in which PAR2 function is important for development. Phenocopies of PAR-deficiencies will help identify which of the several G proteins regulated by PARs is important for the phenotypes described above, and novel phenotypes will point to new roles for G protein-coupled receptors in embryonic development. We also plan systematic candidate-driven approach to identify PAR2-activating proteases that might function in the embryo. These studies will provide new insights into basic mechanisms underlying hematopoiesis, vascular development and/or neurulation.